Rapidly evolving pathogens, such as the human immunodeficiency virus (HIV), escape immune defenses provided by most vaccine-induced antibodies. Proposed strategies to elicit broadly neutralizing antibodies (bnAbs) require a deeper understanding of evolution of the immune response to vaccination or infection. In HIV infected individuals, viruses and B-cells evolve together, creating a virus-antibody arms race. The objective of this proposal is to analyze the arms race in donors such as CH505 and CH0694, who have developed antibodies of significant breadth, which would be informative for immunogen design. Donor CH505 produced a lineage of antibodies, called CH103, which interact with the gp120 CD4 binding site. Analysis of crystal structures of progenitor (UCA) and intermediate Fabs, along with binding measurements to various HIV envelope gp120s, indicated that there was a shift in the relative orientation of the variable light-chain (VL) and heavy-chain (VH) domains during evolution in this lineage to relieve unfavorable contacts with gp120. Because a VH-VL shift would change the way the antibodies interact with gp120, a deeper analysis of VH-VL interface changes and changes in affinity for gp120 will be performed. Crystal structures will be determined of gp120 in complex with the UCA or intermediate Fab I3.2, whose VH-VL orientations differ from that of the mature bnAb, already solved in complex with gp120. Molecular dynamics (MD) simulations will also be done, as a collaboration, of free Fabs and Fab/gp120 complexes, and coupled with additional binding experiments. Antibodies from the DH235 lineage, from the same donor, triggered virus escape mutations that improved binding to CH103 lineage antibodies and therefore could have accelerated affinity maturation in the CH103 lineage. Crystal structures of Fabs of DH235 lineage members will be determined to understand how the DH235 antibodies, which have limited neutralization breadth, differ from CH103 bnAbs despite overlapping epitopes, and to reconstruct the interplay among the two lineages and virus. In addition to rapid mutation, HIV also uses heavy glycosylation to evade the immune system. BnAbs that bind to glycans have been isolated, suggesting that viral carbohydrates can serve as vaccine targets. An early bnAb, called DH175, was identified from donor CH0694 and shown to be PGT-like. While many PGT bnAbs recognize N332 glycosylation, their actual epitopes differ in the glycans and gp120 peptides they recognize. To understand antibody affinity maturation and virus evolution in this lineage, crystal structures of the DH175/gp120 complex and of the free Fabs of precursor antibodies will be determined. Hypotheses concerning stages of affinity maturation deduced from the crystal structures will be tested by introducing mutations into the Fabs and/or gp120 envelope and determining affinities and neutralization properties. MD simulations will also be performed, as a collaboration, if appropriate. These data will identify mechanism(s) that confer broadly neutralizing characteristics on glycan-dependent antibodies.